The Role of Hypoxia-Inducible Factor Isoforms in Breast Cancer and Perspectives on Their Inhibition in Therapy

Round 1

Reviewer 1 Report

In this manuscript, Kozal and Krześlak review hypoxia inducible factors and the research and concepts defining the hypoxic state, regulation of HIF isoforms and the roles and functions of HIF proteins downstream after activation. While these topics have been extensively reviewed by others previously, the authors also focus on the roles of  HIF1a and HIF2a in Breast Cancer and offer a thoughtful perspective on targeting HIFs in that indication. The review is balanced and critiques highlighted studies appropriately, however, I have the following concerns and suggestions:

1) The manuscript is significantly undercited and many of the original research articles that the authors refer to are either not cited appropriately or are attributed to other review papers.   a) Examples of no original research articles cited. -Lines 90-127:  -Lines 139-145:   b) Statements without appropriate citations:   Lines 131-134: "First, the HIF-2α isoform seems more resistant to the hydroxylation maintained by PHDs and FIH and regulations by other factors; hence it shows much higher stability than HIF-1α. The HIF-2α transcriptional activity promotes the activation of the hypoxia-associated factor (HAF) whose activity leads to the ubiquitination of the HIF-1α subunit resulting in its degradation"
Lines 155-159: "A significant percentage (88%) of normal human tissues does not contain HIF-1α due to its quick degradation. Still, it can be detected in over half of the cancers, such as prostate cancer, breast cancer (BC), lung cancer, pancreatic cancer, brain cancer, stomach cancer, ovarian cancer, kidney cancer, and melanomas."
Lines 203-206: "For example, increased activity of the PI3K/Akt/mTOR pathway observed in breast cancer can lead to an increased level of HIF-1α. Although increased signal trans-duction pathways activity is not exclusive to this type of cancer, in BC, overexpression of HER2 or ER receptors may contribute to an increased level of HIF-α through the PI3K/Akt/mTOR pathway."
Lines 231-234: "Extracellular ATP can also drive HIFs’ activation in normoxia via the Akt pathway. The activity of this transduction pathway, induced by the 2presence of the extracellular ATP, results in increased activity of PGK1, which forms a feedforward loop with the HIF-1α isoform, whose activity leads to upregulation of PGK1."
Line 260-261: "The earliest discovered role of the hypoxia-inducible factor was stimulating the formation of new blood cells by inducing the activity of the erythropoietin gene."
Lines 366-369: "Anhydrase 9 plays a critical role in maintaining pH levels. In normal breast tissue, the expression of CA IX is not detected, but it is often expressed in breast cancer. It may be caused by over-expression of HIF-1α which targets the anhydrase’s gene. It is believed that only the HIF-1α isoform contributes to acidification."
Lines 403-413:" BAK, CASP3, CASP8, CASP10, and TNFRSF10A were identified as pro-apoptotic genes whose expression was decreased by HIF-1 in hypoxia when compared to normoxic conditions, in the presence or absence of paclitaxel. HIF-1 was also shown to increase the expression of Mcl-1 and BNIP-3. Mcl-1 (myeloid cell leukemia sequence 1) is an anti-apoptotic protein belonging to the Bcl-2 family, which suppresses cytochrome c release from mitochondria via hetero-dimerization and neutralization of pro-apoptotic proteins such as Bim or Bax. BNIP3 is a member of the Bcl-2 family of mitochondrial proteins, which triggers selective mitochon-drial autophagy. BNIP3 competes with Beclin-1 for binding to Bcl-2, thereby freeing Be-clin-1 to activate autophagy. Stimulation of autophagy might be especially crucial to in-duce cell death in apoptosis-resistant breast cancer cell lines."
Lines 506-510: "CD44 and aldehyde dehydrogenase is the other two critical factors of CSCs. It has been shown that hypoxia can promote the induction and development of CSCs through these two factors in various cancers including breast. High activity of ALDH is associated with stem cell features including self-renewal, metastasis, tumorigenesis, and poor prognosis in breast cancer."
2)  Although individual genes regulated by HIF1a and HIF2a are discussed, the review should include a discussion and synthesis of papers describing genomic and transcriptomic approaches to characterizing HIFs and hypoxia in breast cancer samples and cell lines (i.e. RNAseq/DNAseq/ChIPseq). 

Author Response

• The manuscript is significantly undercited and many of the original research articles that the authors refer to are either not cited appropriately or are attributed to other review papers.

Thank you very much for this comment. Since our manuscript covers many aspects of HIFs function we are not able to cite all valuable papers that certainly should be cited in reviews more focused on one or two function. Moreover, we tried to avoid citing older papers. However, the Reviever’s comments made us to think over our citation choices. That is why we have added over a dozen new references positions.

Lines 90-127: The HIF-1α and HIF-2α isoforms show similar regulation characteristics, although they are active in different types of hypoxia. In molecule characteristics, those isoforms share approximately 48% amino acid sequence identity [8]. The HIF-2α also called the endothelial PAS domain protein (EPAS1), differs from the HIF-1α subunit mainly in the N-terminal transactivation domain (N-TAD), while C-TAD domains share 67% of the similarities Hu, C. J., Sataur, A., Wang, L., Chen, H., & Simon, M. C. (2007). The N-terminal transactivation domain confers target gene specificity of hypoxia-inducible factors HIF-1alpha and HIF-2alpha. Molecular biology of the cell, 18(11), 4528–4542. https://doi.org/10.1091/mbc.e06-05-0419 [9].

  -Lines 139-145:   b) Statements without appropriate citations:   Lines 131-134: "First, the HIF-2α isoform seems more resistant to the hydroxylation maintained by PHDs and FIH and regulations by other factors; hence it shows much higher stability than HIF-1α Holmquist-Mengelbier, L.; Fredlund, E.; Löfstedt, T.; Noguera, R.; Navarro, S.; Nilsson, H.; Pietras, A.;Vallon-Christersson, J.; Borg, Å.; Gradin, K.; et al.  Recruitment of HIF-1αand HIF-2αto common target genes is differentially regulated in neuroblastoma: HIF-2αpromotes an aggressive phenotype.Cancer Cell 2006,10, 413–423, Koivunen, P.; Hirsilä, M.; Günzler, V.; Kivirikko, K.I.; Myllyharju, J. Catalytic Properties of the AsparaginylHydroxylase (FIH) in the Oxygen Sensing Pathway Are Distinct from Those of Its Prolyl 4-Hydroxylases.J. Biol. Chem.2004,279, 9899–9904 . The HIF-2α transcriptional activity promotes the activation of the hypoxia-associated factor (HAF) whose activity leads to the ubiquitination of the HIF-1α subunit resulting in its degradation" Koh, M.Y.; Darnay, B.G.; Powis, G. Hypoxia-Associated Factor, a Novel E3-Ubiquitin Ligase, Binds and Ubiquitinates Hypoxia-Inducible Factor 1alpha, Leading to Its oxyden-indepedent degradation.Mol CellBiol 2008,28, 7081–7095

Lines 155-159: "A significant percentage (88%) of normal human tissues does not contain HIF-1α due to its quick degradation. Still, it can be detected in over half of the cancers, such as prostate cancer, breast cancer (BC), lung cancer, pancreatic cancer, brain cancer, stomach cancer, ovarian cancer, kidney cancer, and melanomas." Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Activation of the HIFpathway in cancer. Curr Opin Genet Dev 11(3):293–299

 

Lines 203-206: "For example, increased activity of the PI3K/Akt/mTOR pathway observed in breast cancer can lead to an increased level of HIF-1α. Although increased signal trans-duction pathways activity is not exclusive to this type of cancer, in BC, overexpression of HER2 or ER receptors may contribute to an increased level of HIF-α through the PI3K/Akt/mTOR pathway." Semenza, Gregg L. “HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations.” The Journal of clinical investigation vol. 123,9 (2013): 3664-71. doi:10.1172/JCI67230

Lines 231-234: "Extracellular ATP can also drive HIFs’ activation in normoxia via the Akt pathway (Shi YH, Wang YX, Bingle L, et al. In vitro study of HIF‐1 activation and VEGF release by bFGF in the T47D breast cancer cell line under normoxic conditions: involvement of PI‐3K/Akt and MEK1/ERK pathways. J Pathol. 2005;205:530‐536.). The activity of this transduction pathway, induced by the presence of the extracellular ATP, results in increased activity of PGK1, which forms a feedforward loop with the HIF-1α isoform, whose activity leads to upregulation of PGK1.Fu D, He C, Wei J, et al. PGK1 is a potential survival biomarker and invasion promoter by regulating the HIF‐1α‐mediated epithelial‐mesenchymal transition process in breast cancer. Cell Physiol Biochem. 2018;51:2434‐2444."

Line 260-261: "The earliest discovered role of the hypoxia-inducible factor was stimulating the formation of new blood cells by inducing the activity of the erythropoietin gene." Wang, G L, and G L Semenza. “General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.” Proceedings of the National Academy of Sciences of the United States of America vol. 90,9 (1993): 4304-8. doi:10.1073/pnas.90.9.4304

Lines 366-369: "Anhydrase 9 plays a critical role in maintaining pH levels. In normal breast tissue, the expression of CA IX is not detected, but it is often expressed in breast cancer Lock FE, McDonald PC, Lou Y, Serrano I, Chafe SC, Ostlund C, et al Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche. Oncogene 2013;32:5210–9. It may be caused by over-expression of HIF-1α which targets the anhydrase’s gene. It is believed that only the HIF-1α isoform contributes to acidification Moreno Roig, Eloy et al. “HIF-1α and HIF-2α Differently Regulate the Radiation Sensitivity of NSCLC Cells.” Cells vol. 8,1 45. 12 Jan. 2019, doi:10.3390/cells8010045."

Lines 403-413:" BAK, CASP3, CASP8, CASP10, and TNFRSF10A were identified as pro-apoptotic genes whose expression was decreased by HIF-1 in hypoxia when compared to normoxic conditions, in the presence or absence of paclitaxel Flamant, L., Notte, A., Ninane, N., Raes, M., & Michiels, C. (2010). Anti-apoptotic role of HIF-1 and AP-1 in paclitaxel exposed breast cancer cells under hypoxia. Molecular cancer, 9, 191. https://doi.org/10.1186/1476-4598-9-191. HIF-1 was also shown to increase the expression of Mcl-1 and BNIP-3. Mcl-1 (myeloid cell leukemia sequence 1) is an anti-apoptotic protein belonging to the Bcl-2 family, which suppresses cytochrome c release from mitochondria via hetero-dimerization and neutralization of pro-apoptotic proteins such as Bim or Bax. Michels J, Johnson PW, Packham G. Mcl-1. Int J Biochem Cell Biol. 2005;37(2):267-271. doi:10.1016/j.biocel.2004.04.007 BNIP3 is a member of the Bcl-2 family of mitochondrial proteins, which triggers selective mitochondrial autophagy. BNIP3 competes with Beclin-1 for binding to Bcl-2, thereby freeing Be-clin-1 to activate autophagy Zhang, H., Bosch-Marce, M., Shimoda, L. A., Tan, Y. S., Baek, J. H., Wesley, J. B., Gonzalez, F. J., & Semenza, G. L. (2008). Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. The Journal of biological chemistry, 283(16), 10892–10903.. Stimulation of autophagy might be especially crucial to in-duce cell death in apoptosis-resistant breast cancer cell lines. Dalby KN, Tekedereli I, Lopez-Berestein G, Ozpolat B (2010) Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 6(3):322–329. PubMed PMID: 20224296; PubMed Central PMCID: PMCPMC2914492"

Lines 506-510: "CD44 and aldehyde dehydrogenase is the other two critical factors of CSCs. It has been shown that hypoxia can promote the induction and development of CSCs through these two factors in various cancers including breast Ricardo, S., Vieira, A. F., Gerhard, R., Leitão, D., Pinto, R., Cameselle-Teijeiro, J. F., Milanezi, F., Schmitt, F., & Paredes, J. (2011). Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. Journal of clinical pathology, 64(11), 937–946. https://doi.org/10.1136/jcp.2011.090456. High activity of ALDH is associated with stem cell features including self-renewal, metastasis, tumorigenesis, and poor prognosis in breast cancer Ginestier, C., Hur, M. H., Charafe-Jauffret, E., Monville, F., Dutcher, J., Brown, M., Jacquemier, J., Viens, P., Kleer, C. G., Liu, S., Schott, A., Hayes, D., Birnbaum, D., Wicha, M. S., & Dontu, G. (2007). ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell, 1(5), 555–567. https://doi.org/10.1016/j.stem.2007.08.014."

• Although individual genes regulated by HIF1a and HIF2a are discussed, the review should include a discussion and synthesis of papers describing genomic and transcriptomic approaches to characterizing HIFs and hypoxia in breast cancer samples and cell lines (i.e. RNAseq/DNAseq/ChIPseq).

 

We agree that synthesis of papers describing genomic and trancriptomic approaches to characterize HIFs in breast cancer would be valuable and could be potentially interesting for readers. However, we think that this is a little bit beyond the scope of our paper. In our manuscript we tried to analyze and present all data that could help us to answer the question whether HIFs’ inhibition in breast cancer treatment is worth the effort. The research methodology was not the aspect we were focused on. Still, we think that this is a very interesting topic to discuss in future papers.

Reviewer 2 Report

I would first acknowledge authors for putting together an amazing work with respect to role of isoforms of hypoxia inducible factors isoforms during tumor progression. Its a very well written manuscript and provide great insights in the field.

1. I agree with authors that targeting hypoxia inducible factors in clinical trials may be way to address hallmarks of tumor progression. On the same line, I do have a suggestion, to include a section of clinical trials with agents targeting hypoxia inducible factors or molecules in the pathway and also providing concise tabular data as well. 

2. Additionally, I would suggest adding some figures to the manuscript. In the current format, it is mostly driven by what is known in the literature, I would be interested in a figure summarizing the mechanistic insights of different isoforms of hypoxia and how they regulate tumor progression. 

Author Response

1. I agree with authors that targeting hypoxia inducible factors in clinical trials may be way to address hallmarks of tumor progression. On the same line, I do have a suggestion, to include a section of clinical trials with agents targeting hypoxia inducible factors or molecules in the pathway and also providing concise tabular data as well.

 

In the course of working on the first version of the manuscript, we prepared the table with data from clinical trials. There are several trials with published preliminary results.  However,  most ongoing trials on HIFs inhibitors do not apply to breast cancer.  That is why we decided not to include the table. The most advanced and interesting data concern studies on renal cancer and we mentioned them in the last section of our manuscript. Data concerning the progress of HIF inhibition in clinical trials may be found in the paper :  Fallah, J., Rini, B.I. HIF Inhibitors: Status of Current Clinical Development. Curr Oncol Rep 21, 6 (2019). https://doi.org/10.1007/s11912-019-0752-z

 

 

 

2. Additionally, I would suggest adding some figures to the manuscript. In the current format, it is mostly driven by what is known in the literature, I would be interested in a figure summarizing the mechanistic insights of different isoforms of hypoxia and how they regulate tumor progression.

Thank you very much for this comment. According to the suggestion, we have added a new Figure that summarizes the information concerning similarities and differences between HIFs’ isoforms function in breast cancer.

Reviewer 3 Report

In this manuscript, the authors reviewed the role, functions, and perspectives on hypoxia-inducible factors inhibition in breast cancer. Their work could be of great value for a better understanding antitumor effect and the development of novel drugs for cancer treatment. The well written manuscript requires no changes or editing.

Author Response

Thank you very much

Round 2

Reviewer 1 Report

Thank you for addressing my concerns.